Protein is of a stereoscopic conformation called as higher-order structure, and it is essential for function of a protein to be of a correct higher-order structure. Of these higher-order structures, α-helical structure, β-sheet structure etc. are known as secondary structures, and the protein of thus mentioned secondary structure is further folded to form a tertiary and quaternary structure. As factors for stabilizing the tertiary structure, hydrophobic bond, hydrogen bond, S—S bond between cysteine residues etc. are included.
This higher-order structure is destroyed by physical causes such as heating, freezing, ultraviolet radiation and irradiation of X-ray, as well as by chemical causes such as extreme acid and base, organic solvent, denaturant such as urea and guanidine hydrochloride, and detergent, and converts from orderly folded compact structure to a random coil which is unfold state.
Folding in which the protein of above-mentioned denatured state is refolded to the condition having a correct higher-order structure has two stepwise problems which must be solved. That is, the first step problem is to prevent aggregation between proteins, and the second step problem is how to fold correctly from unfolded state.
In an organism, a class of second (or assistant) proteins a named as molecular chaperon participates in these two steps. Molecular chaperon is a protein having functions of binding to a protein just synthesized to stop misfolding to keep the protein in the state that it is easily carried, and helping the protein which can not be folded well to be folded.
Recently, a trial is reported aiming at constructing an artificial chaperon which mimics a molecular chaperon to solve above two problems.
Daugherty et al. (J. Biol. Chem., vol. 273, No. 51, p.33961-33971 (1998)) has reported a method for refolding a denatured protein into a native structure by using Triton X-100 or polyoxyethylenic detergent having a short chain length of alkyl group detergent, as an artificial chaperon. These two nonionic detergents play a role to prevent proteins from aggregation, and thereafter are removed from the protein-detergent complex in second step.
In this method, β-cyclodextrin (hereinafter, sometimes abbreviated as β-CD), a cyclic saccharide which can accommodate various guest molecules into the hydrophobic cavity, is used as a second (or assistant) agent to strip detergent from protein-detergent complex.
Further, Sivakama Sundari et al. (FEBS Letters, 443, p.215-219 (1999)) shows a method for correctly folding the denatured protein by diluting denatured carbonic anhydrase B (hereinafter, sometimes abbreviated as CAB) and lysozyme with cetyltrimethylammonium bromide (hereinafter, sometimes abbreviated as CTAB) which is a cationic detergent, thereafter acting straight chain dextrin-10 or cyclic β-CD.
However, in any method mentioned above, the ratio in which denatured inactive protein is refolded into the native form is only about 60 to 70%, and β-CD has a problem concerning stability that, such as, low solubility in water and unstableness of solution because of aging, and is not satisfactory for using as a perfect artificial chaperon.
Also, there has been a trial for refolding a denatured protein by dilution dialysis method wherein a denaturant is slowly removed from the protein in denatured state, but applicable proteins are limited: to those having a voluntary folding ability such that the protein spontaneously refolds to the native form even without any special treatment.
Thus, it is an actuality that all of the above-mentioned trials have a problem in that the ratio that the protein is folded as the native form is low, and that the trials can not be applied to the proteins which have a low voluntary folding ability and can not be of a native form without a second (or assistant) of a molecular chaperon.